Random mutagenesis of a plasmid-borne glycosidase gene and phenotypic selection of mutants in Escherichia coli.

The bglA gene from Bacillus polymyxa encodes a beta-glucosidase able to hydrolyze p-nitrophenyl-beta-D-glucopyranoside (PNPG), and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal), chromogenic substrates for beta-glucosidases and beta-galactosidases respectively. A plasmid carrying the blgA gene inserted in vector pUC18 was mutagenized in vitro with hydroxylamine, and subsequently used to transform E. coli selecting for the ampicillin resistance conferred by the cloning vector. The transformants were screened on petri dishes for mutations causing inability to hydrolyze either one of the two substrates, and for mutations increasing resistance of the enzyme to thermal inactivation. The isolation of several mutants with such characteristics suggests that the simple procedure used here can be applied to generate modifications of enzymatic properties that fit specific industrial requirements.

High and low molecular weight heparins do not modify red blood cell aggregability in vitro.

The usefulness of heparins as anticoagulants has been demonstrated, but their effects on hemorheological parameters, such as erythrocyte aggregability, are under discussion. If the heparins had adverse effect on erythrocyte aggregability, its use would be especially undesirable in patients with pathologies involving red blood cell hyperaggregability as is the case of cardiac disease. In the present study we examine the in vitro effect of unfractionated and fractionated heparins on red blood cell aggregability. The results show that heparins do not increase this rheological parameter but show a slight tendency to lower it.

Structural basis of increased resistance to thermal denaturation induced by single amino acid substitution in the sequence of beta-glucosidase A from Bacillus polymyxa.

The increasing development of the biotechnology industry demands the design of enzymes suitable to be used in conditions that often require broad resistance against adverse conditions. beta-glucosidase A from Bacillus polymyxa is an interesting model for studies of protein engineering. This is a well-characterized enzyme, belonging to glycosyl hydrolase family 1. Its natural substrate is cellobiose, but is also active against various artificial substrates. In its native state has an octameric structure. Its subunit conserves the general (alpha/beta)8 barrel topology of its family, with the active site being in a cavity defined along the axis of the barrel. Using random-mutagenesis, we have identified several mutations enhancing its stability and it was found that one them, the E96K substitution, involved structural changes. The crystal structure of this mutant has been determined by X-ray diffraction and compared with the native structure. The only difference founded between both structures is a new ion pair linking Lys96 introduced at the N-terminus of helix alpha2, to Asp28, located in one of the loops surrounding the active-site cavity. The new ion pair binds two segments of the chain that are distant in sequence and, therefore, this favorable interaction must exert a determinant influence in stabilizing the tertiary structure. Furthermore, analysis of the crystallographic isotropic temperature factors reveals that, as a direct consequence of the introduced ion pair, an unexpected decreased mobility of secondary structure units of the barrel which are proximal to the site of mutation is observed. However, this effect is observed only in the surrounding of one of the partners forming the salt bridge and not around the other. These results show that far-reaching effects can be achieved by a single amino acid replacement within the protein structure. Consequently, the identification and combination of a few single substitutions affecting stability may be sufficient to obtain a highly resistant enzyme, suitable to be used under extreme conditions.

Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A.

Mutations enhancing the thermostability of beta-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed beta-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while their kinetic parameters did not change. CD spectra indicated that the E96K replacement caused an increase in alpha-helix content. The observed effect of the M416I mutation is consistent with the lower content of cysteine and methionine found in family 1 enzymes of thermophilic species compared with similar ones from mesophilic organisms.